Journal: Nucleic Acids Research

Article Title: NUP98 regulates orthoflavivirus replication through interaction with vRNA and can be targeted for antiviral purposes

doi: 10.1093/nar/gkag027

Figure Lengend Snippet: NUP98 interacts with an RNA motif at the end of the E coding region. ( A ) Experimental layout of CLIP assay. CLIP assay of HEK293T cells transfected with eGFP-NUP98 or eGFP-NUP98[1–729] infected with LGTV MOI 1 for 48 h. Immunoprecipitation performed using anti-eGFP antibody (triangle) or control beads (square). cDNA performed using random primers ( B ) or strand-specific viral primers ( C ). Values were normalized to input vRNA and control bead precipitated samples for each condition. n = 9. Significance calculated using one-way ANOVA with Tukey’s multiple comparison test. **** < 0.0001. ( D ) Focus forming assay of 24 h TBEV (MOI 0.1) infected CRISPR NUP98 KD HEK293T cells transfected with eGFP, eGFP-NUP98, and eGFP-NUP98[1–729]. n = 10. Bars represent mean + SEM. Significance calculated with two-way ANOVA with Fisher’s LSD test. * < 0.05; ** < 0.01. ( E ) Schematic representation of NUP98. NRM, nucleoporin RNA-binding motif; GLEBS, gle2-biding sequence; GLFG, glycine–leucine–phenylalanine–glycine repeats; NUP98-Frag., purified NUP98 construct used in this study. ( F ) SPR experiment showing binding of purified NUP98–CTD. with different in vitro transcribed RNA fragments in different concentrations (31.25 nM–62.5 nM–125 nM–250 nM–500 nM). The Y-axis indicates RU. n = 3. The Bars represent means + SD. ( G ) CLIP-seq experiment on HEK293T cells transfected with eGFP-NUP98 infected with TBEV MOI 1 48 h. Mapping of positive- and negative-strand RNA reads to the TBEV genome. Two replicates are shown. Significant peaks of bound positive-strand RNA are depicted in red boxes. ( H, I ) RNA electrophoretic mobility shift assay (RNA EMSA) of Cy3-tagged vRNA [2119–2264 nt] or NS2B encoding vRNA and indicated concentrations of NUP98–CTD or GST protein. ( H ) The bound fraction was plotted against the NUP98–CTD concentration ( n = 3; mean ± SD). K D and curves were determined with nonlinear one site-specific binding equation.

Article Snippet: The RNA was then extracted and used to prepare a cDNA library using the NEBNext small RNA sequencing kit (New England Biolabs) according to manufacturer’s instructions.

Techniques: Transfection, Infection, Immunoprecipitation, Control, Comparison, Focus Forming Assay, CRISPR, RNA Binding Assay, Sequencing, Purification, Construct, Binding Assay, In Vitro, Electrophoretic Mobility Shift Assay, Concentration Assay

Journal: Journal of Advanced Research

Article Title: Senkyunolide A interrupts TRAF6-HDAC3 interaction to epigenetically suppress c-MYC and attenuate cholestatic liver injury

doi: 10.1016/j.jare.2025.04.002

Figure Lengend Snippet: RNA-sequencing analysis and experimental verification of the inhibitory effects of SenA on hyperproliferated cholangiocytes. (a) Flowchart of mouse primary cholangiocytes isolation with associated photographic images. (b) GO analysis of DEGs between BDL vs . Sham and DEGs between BDL + SenA vs BDL by R software. The size of dots was used to describe the number of genes enriched in corresponding pathways and the color of dots indicates the significance. (c) The relative expression levels of DEGs implicated in cellular proliferation were shown as a heatmap. (d) Relative mRNA levels of Pcna , Epcam , Myc and Sphk2 in mouse primary cholangiocytes. Hprt1 was utilized as an internal control (n = 3). (e) Representative images of CK7 and KI67 co-immunofluorescence staining. (f) The cell cycle was detected by PI staining. Relative mRNA levels of (g) PCNA and SPHK2 and (h) P21 and P16 in HIBECs. Representative immunoblots against (i) PCNA and SPHK2 and (j) P21 and P16 and β-ACTIN in HIBECs were shown. * P < 0.05, *** P < 0.001, vs. Control group; # P < 0.05, ## P < 0.01, ### P < 0.001, vs. Model group (n = 3).

Article Snippet: The library fragments (250–300 bp) were purified with the AMPure XP system (Beckman Coulter, USA) and sequencing library was established by NEBNext UItra TM RNA Library Prep Kit for Illumina (NEB) .

Techniques: RNA Sequencing, Isolation, Software, Expressing, Control, Immunofluorescence, Staining, Western Blot